A wound inducible expression construct and a method of its preparation

ABSTRACT

The present invention provides a wound inducible expression construct and a method of its preparation. The invention provides a methods for isolation of an early wound inducible promoter that is activated within 5 minutes of any form of wounding (mechanical or biological) and a process of making transgenic plants in which expression of GUS/Insecticidal protein is regulated by Promoter (I.D.1) in a wound inducible manner for local expression of a chimeric gene used in this method and plants obtained thereby, and to the process for obtaining resistance to insect feeding.

FIELD OF THE INVENTION

The present invention relates to a wound inducible expression constructand a method of producing plants in which expression of any availablenatural or commercial gene, which can be translated in to a functionalprotein, such as but not restricted to GUS or an insecticidal protein invery short time. This expression is regulated by an early novel woundinducible RbPCD1 (Rosa bourboniana) promoter of sequence I.D.1.Invention further relates to identification and isolation of this novelpromoter RbPCD1 by using a set of forward primer GSP1 and reverse primerGSP2 of sequence I.D.2 and I.D.3 respectively as given below.

GSP1 5′-TAACCGCTAGGCAGTGAGC-3′ GSP2 5′-CTTCTTCTCTGTTACCTGAAA-3′

PRIOR ART OR BACKGROUND ART

Plant productivity is affected by several factors, one of the major onesbeing insect damage. Both chewing and sucking pests feed on plantleaves, shoots, roots, fruits, flowers and grains leading toconsiderable loss of yield. Insect infestation is one of the majorconcerns for crop production. In this regard, it is necessary to developnovel approaches to reduce crop losses to insects and thereby increasenet yield. Genetic transformation is a powerful tool for production ofcrop plants with increased resistance to phytopathogens. A number oftransgenic cultivars with elevated tolerance to economically importantpests and disease agents are in commercial production. However, in mostof these the transgene is driven by a powerful constitutive promoter,such as the cauliflower mosaic virus 35S (CaMV 35S) promoter and itsderivatives, and is expressed at high levels even in the absence ofpathogen invasion. Continuous synthesis and high accumulation oftransgene products, especially toxins, could interfere with plantmetabolic pathways and the overall expression of other valuable traitsand reduce yields. The above mentioned strategies, although effective,are associated with several problems that affect yield such as:

-   -   Abnormalities in plant growth (leading to yield penalty) due to        high level expression of the toxic proteins which even affect        normal plant development (Barton et al., 1987; Diehn et al.,        1996; Rocher et al., 1998; Sachs et al., 1998; Rawat et al.,        2011).    -   Reduced frequency of transgene incorporation in many plants (due        to high levels of toxic protein that affect regeneration) as a        result of which a much larger number of calli have to be created        to get the requisite number of transformants (Rawat et al.,        2011).    -   Reduced expression of insecticidal protein at times of flowering        (especially with the most commonly used CaMV35S promoter, which        shows reduced expression at the time of flowering) in plants        like cotton and maize leading to susceptibility to insect attack        during flowering and boll formation by boll worm which reduces        yields (Kranthi et al., 2005).    -   Diversion of vital plant resources towards maintaining high        levels of toxic protein even when they are not needed.

In contrast, the use of promoters of plant defensive genes has distinctadvantages because most of them are activated only when the plant isattacked by pests or pathogens. The use of native plant promoters canalso help to avoid transgene silencing often associated with thepresence of promoters of non-plant origin in the plant genome andparticularly the CaMV35S promoter [Matzke et al., Plant Physiol. 107(1995) 679-685]. Plants have developed a variety of physical andbiochemical defense barriers against pests and pathogens. Mechanicalwounding of plant tissue (mimicking pathogen invasion or insect chewing)leads to the accumulation of mRNAs that encode proteins thought to beinvolved in plant defense [Bowles, Annu. Rev. Biochem. 59 (1990)873-907], and provides a convenient system to isolate and studydefense-related genes and their upstream regulatory regions intransgenic host. Some of the promoters developed include the AoPR1promoter from Asparagus that is activated in 6 hours (Warner et al.,1993; Gulbitti-Onarici et al., 2009), the Shpx6B promoter fromStylosanthes humili speroxidise gene which is activated in 24-48 hoursafter insect feeding (Perrera and Jones, 2004). However, the time takenfor activation of these promoters (6-48 hours) is rather longconsidering that insects take a much shorter time to feed on andinitiate damage on plants. Thus identification of strong wound induciblepromoters that express target proteins rapidly only at the time ofwounding and their utilization for expression of insecticidal proteinsis desirable.

Limitation in Prior Art

Presently no wound inducible promoter has been identified that isactivated within a very short time (5-20 minutes) of wounding for rapidsynthesis of any protein or chemical and that can act against both typesof insects (chewing as well as sucking). Hence there was need to isolatewound specific promoter in plants, which gets activated quickly andpreferably in 5-20 minutes.

OBJECTIVES

The main objective of the present invention is to provide a woundinducible expression construct and a method to develop transgenic plantswhich show quick response to wounds caused by biological/mechanicalreasons. Here quick response means expression of gene under control ofnovel promoter RbPCD1 within 5-20 minutes.

Another objective of the invention is to develop insect resistanttransgenic plants having a strong mechanical/biological wound induciblepromoter which is isolated by synthetically designed primers that allowshigh level expression of the target protein within 5-20 minutes ofwounding (by insects and other biotic pests).

Another objective of the invention is to develop method for developinginsect resistant plants by using plant expression vector having RbPCD1promoter of sequence I.D.1.

Yet another objective of the invention is to develop a method ofidentification and isolation of wound inducible promoter.

Yet another objective of the invention is to develop a method to studythe tissue specificity of the promoter in presence and absence ofwounding and during normal plant growth.

Yet another objective of the invention is to develop a method tovalidate the wound induction of the promoter in diverse plant familiesof economic importance representing both monocot and dicot kingdoms foruniversality of expression and use.

Yet another objective of invention to have transgenic plants having apromoter of sequence I.D.1 with different insecticidal toxic genes thatare effective against both chewing and sucking pests.

Yet another objective of the invention is to express any gene and obtainhigh levels of any protein or enzyme within 5-20 minutes using thepromoter with sequence ID 1.

Yet another objective is to test the efficacy of the promoter byexpression of an insecticidal toxin protein under its control in a woundinducible manner.

Yet another objective to have transgenic plants with controlledexpression of insecticidal gene only under wounding to ensure normalplant development and to allow several other plants which are currentlynot so easily transformed due to insect protein toxicity to betransformed.

Yet another objective is to develop a process for reduced load on theplant by synthesizing the toxic protein only at the time of insectinfestation/wounding and not at all times when it is not needed.

Yet another objective is to develop a process for construct preparationunder control wound inducible promoter.

Yet another objective to develop a method of Agrobacterium mediatedplant transformation of the above said construct.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 A schematic presentation of the RbPCD1 promoter construct forplant transformation. Restriction sites relevant for cloning are initalics (SalI and HindIII). (A) Construct (pBI101-RbPCD1pro::GUS) forGUS gene expression under RbPCD1 promoter of sequence I.D.1 cloned atSalI restriction enzyme site (B) Construct (pBI101-RbPCD1pro::Cry1Ac)for cry1Ac gene expression under RbPCD1 promoter of sequence I.D.1cloned at HindIII restriction enzyme site.

FIG. 2. Illustrates the histochemical analysis of GUS expression ofRbPCD1promoter of sequence I.D.1 and pBI121 vector as control. (A)represents 5 min, 20 min Arabidopsis wounded leaf (Colour developmentwas carried out for 3 h in presence of cycloheximide), (B) representsGUS activity in Helicoverpa armigera and Aphid fed transgenicArabidopsis lines (C) GUS activity in pBI121 vector transformedArabidopsis, chickpea transgenic lines and agroinfiltrated rose petal(D) Agro-infiltration and histochemical GUS analysis ofRbPCD1promoter-GUS in rose petals, cotton sepal, gladiolus tepal andtobacco leaves and in stable transgenic shoots of chickpea afterwounding. (E) GUS activity during plant development in Arabidopsistransgenic lines.

FIGS. 3.(A) & (B) Detached leaf insect bioassay of Arabidopsis andtomato transgenic lines expressing Cry1Ac protein under RbPCD1 promoterof sequence I.D.1 fed upon by the insect Helicoverpa armigera atdifferent stages of growth.

FIGS. 4.(A) & (B) Insect bioassay of Arabidopsis and tomato transgeniclines expressing Cry1Ac protein under RbPCD1 promoter with 3^(rd) instarlarva of the insect Helicoverpa armigera on intact plant leaf.

FIG. 5 Table represents comparative expression of Cry1Ac protein underRbPCD1 promoter and CaMV35S constitutive promoter measured by ELISA.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides an early wound induciblepromoter which is activated in a very short time interval (5-20 minutes)by any form of wounding (mechanical or biological) using novelsynthetically designed sets of primers of sequence I.D.2 and 3 from Rosefor making transgenic plants having desirable gene under control of thispromoter. This gene may be insecticidal protein encoding genes but notrestricted to it.

Further the present invention relates to a method for identification andisolation of a strong wound inducible promoter of sequence I.D.1 whichis activated within a very short time interval (5-20 minutes) aftermechanical/biological wounding. A particular embodiment of presentinvention, relates to a method for obtaining early wound inducedexpression of desired proteins such as GUS/Cry proteins inmonocotyledonous as well as dicotyledonous plants. The invention alsorelates to development of insect resistant plants against chewing orsucking type of insects. The present invention provides expression of aninsecticidal protein or any desired protein in a wound inducible manner.In one embodiment a DNA sequence that represents the proximal promoterand 5′ untranslated region of the programmed cell death like gene(RbPCD1) from the fragrant variety of rose (Rosa bourboniana) isisolated using the primer set of sequence I.D.2 and I.D.3. The DNAsequence of 523 nucleotides of sequence I.D.1 can drive the expressionof any target gene situated downstream of the promoter at a high levelin a wound inducible manner. The promoter is activated and leads to highlevel accumulation of the target protein within 5-20 minutes in responseto any form of mechanical wounding (such as by a pin or forceps) or byexcision of the leaf or stem tissue with a blade or by wounding causedby insect chewing or sucking or by tissue abrasion. Non significant orno expression is observed in absence of any form of wounding.

Transgenic plants like but not restricted to Arabidopsis, Tomato,Chickpea have been developed using a plant expressing vector havingRbPCD1 promoter of sequence I.D.1 and demonstrated to act in theseplants. It has also been demonstrated to act in plants like Rose,Cotton, Tobacco, and Gladiolus when it was introduced throughagro-infiltration into these plants.

DETAILED DESCRIPTION In the Description of Present Invention, Terms Usedhas been Defined Below

Gene and Promoter: The term “gene” as used herein refers to segments ofDNA located on genome and it contains the codes for the production ofspecific proteins. A gene consists of a long combination of fourdifferent nucleotide bases (chemicals) that are: A, T, C, and G. Any 3nucleotides out of four make a code for amino acid called codon (withthe exception of TAG, TGA and TAA). There are many possible combinationsof four nucleotides. Typically in any plant cell, the 5′UTR (5′untranslated region), the coding region and the 3′UTR (3′ untranslatedregion) are transcribed into RNA by a process called transcription(regulated by a sequence of regulatory region of DNA called promoter),of which, in case of a protein coding gene, the coding region istranslated into protein. A gene may also have an additional sequencecalled intron (non coding region) that is removed prior to translationto a protein. In addition, every gene has a different promoterconsisting of a long set of nucleotides upstream of the 5′UTR that isnot translated but is required to control the expression of the gene ofinterest. In the present case the promoter region is required in a gene(cry1Ac) used for transformation in the current invention.

Chimeric Gene: The term “chimeric gene” refers to any artificial geneconstructed by juxtaposition of fragments of unrelated genes or otherDNA segments, which may themselves have been altered and that are notnaturally associated with each other and/or originate, for example incurrent invention RbPCD1 and cry1Ac both are from different sources.

Genome: A term “genome” of a plant refers to the order of genes and DNAsequences (promoters) in a haploid set of chromosomes.

Foreign nucleic acid: The term “presence of a suitable foreign nucleicacid molecule” as used herein refers to any foreign nucleic acidmolecule that is present in cells of said transgenic plant but absentfrom the cells of the corresponding source plant (Arabidopsis/Tomato).Thereby encompassed are nucleic acid molecules, e.g. gene sequences,which differ from a corresponding nucleic acid molecule in the sourceplant cell. Furthermore encompassed by the term “foreign” are nucleicacid molecules which are homologous with respect to the source plantcell but are situated in a different chromosomal location or differ,e.g., by way of a reversed orientation for instance with respect to thepromoter. In principle, the nucleic acid molecule to be introduced inaccordance with the present embodiment may be of any conceivable origin.It may be from any organism which comprises such molecules. Furthermore,it may be synthetic or derived from naturally occurring molecules by,e.g., modification of its sequence, i.e. it may be a variant orderivative of a naturally occurring molecule. It is, e.g., possible tochange the sequence of a naturally occurring molecule so as to match thepreferred codon usage of plants, in particular of those plants in whichthe nucleic acid molecule shall be expressed.

Induced expression: The term “induced expression” refers to a situationwhere gene expression is obtained or increased by a physical treatment,treatment with a chemical compound or exposure to environmental stimuli.In the current invention the exposure relates to mechanicalwounding/biological wounding.

Insecticidal: “Insecticidal” is used herein to mean a substance toxic toinsects that attack crops. In the current invention the Cry1Ac proteinencoded by the cryIAc gene is used as the insecticidal protein.

Complementary Sequence: Nucleic acid base sequences that can form adouble-stranded structure by matching base pairs; the complementarysequence to G-T-A-C is C-A-T-G.

RbPCD1: This represents abbreviation of a Rosa bourboniana programmecell death like gene and promoter.

Expression vector: An expression vector, otherwise known as anexpression construct, is usually a plasmid, phagemid or virus designedfor protein expression in cells (Plant or Bacterial).

Expression cassette: An expression cassette is a part of a vector DNAused for cloning and transformation. In each successful transformation,the expression cassette directs the cells' machinery to make RNA andprotein. An expression cassette is composed of one or more genes and thesequences controlling their expression. Three components comprise anexpression cassette: a promoter sequence (in current invention, RbPCD1promoter), an open reading frame (in current invention, cry1Ac or GUSgene), and a 3′ untranslated region that, in eukaryotes, usuallycontains a polyadenylation site.

Biotic and abiotic wounding: Biotic wounding is a stress that occurs asa result of damage done to plants by other living organisms, such asherbivores including insects, nematodes, arthropods, fungi, bacteria,viruses etc on the other hand abiotic wounding is stress that occurs asa result of damage done to plants by other non living means includingcold, heat, puncture or scratch made by scalpel, blade forceps etc.

Most of the scientific terms are defined, rest all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which the compositionsand methods described herein belong.

In an embodiment of the present invention it provides a wound inducibleexpression construct consisting of the nucleotide sequence of SequenceID No. 1 or the complementary sequence of it along with the gene ofinterest useful for early activation of wound inducible promoter.

In an embodiment of the present invention it provides a construct wherein the gene of interest is selected from the group comprising ofinsecticidal protein encoding genes against sucking and chewing pestslike cryIAc (and various crystal protein genes), anti-fungal genes likechitinases, genes encoding enzymes that synthesize low levels ofmedicinal compounds in plants.

In an embodiment of the present invention it provides a method forpreparing the construct comprising of steps:

-   -   i. providing RbPCD1 gene having seq id no. 1;    -   ii. amplifying seq id no. 1 using primers selected from, but not        restricted to, GSP 1, and GSP 2 having Sequence ID No. 2, and        Sequence ID No. 3;    -   iii. ligating the gene of interest downstream to the promoter;    -   iv. cloning the sequence obtained in step (iii) in suitable        vector for plant expression

In an embodiment of the present invention it provides a wound induciblepromoter wherein the promoter gets activated within a very short time(5-20 minutes) of wounding.

In an embodiment of the present invention it provides a process formaking transgenic plants comprising wound inducible promoter containingconstruct wherein the process comprises transforming the expressionconstruct into the plant using Agrobacterium mediated transformation;

In an embodiment of the present invention it provides the processwherein the gene of interest is selected from the group comprising ofthose encoding insecticidal proteins, enzymes, antibodies, therapeuticproteins or any protein responsible for producing a valuable chemicalcomprising wounding of transgenic plants.

The present invention provides transgenic plants with the ability toexpress any desired gene within a very short time of mechanical orbiological wounding. This expression is due to the wound inducible DNAsequence of 523 nucleotides of sequence I.D.1, representing the partialupstream portion of the programmed cell death like gene (RbPCD1)promoter and including the 5′ untranslated region (UTR) isolated fromrose using the novel designed synthetic sets of primers of SequenceI.D.2 and I.D.3. The isolated DNA sequence of 523 nucleotides acting aspromoter, can drive the expression of any target gene situateddownstream of the promoter at a high level in a wound inducible manner.This promoter of sequence I.D.1 is activated and leads to high levelaccumulation of the target protein within very short time (5-20 minutes)in response to any form of mechanical wounding (such as by a pin orforceps) or by excision of the leaf/stem tissue with a blade or bywounding caused by insect chewing or sucking or by tissue abrasion. Verylittle or no expression is observed in absence of wounding. In oneembodiment, the amplified fragment was sequenced and cloned into pBI101(a promoter less binary vector) upstream of the beta glucuronidase geneat the SalI site. In another embodiment the chimeric fragment (cry1Acgene under control of wound inducible RbPCD1 promoter) was cloned in toanother pBI101 at the HindIII and EcoRI restriction sites in order tocreate a plant transformation construct. In another embodiment of thepresent invention, the wound inducible construct was introduced intoAgrobacterium tumefaciens by freeze thaw and electroporation methodsrespectively. Agrobacterium cells containing the wound inducibleconstruct was grown in a culture flask and Arabidopsis, tomato andchickpea plants were transformed by floral dip, Agrobacterium mediatedgenetic transformation using leaf disc and cotyledonary methodsrespectively using this construct. Several independent transgenic plantswere raised and screened for the transgene by polymerase chain reactionas well as by their ability to grow on kanamycin (used as a selectionantibiotic to remove untransformed plants). Transgenic plants were grownto T₁, T₂ and T₃ generations by self crossing in case of Arabidopsiswhile tomato plants were grown to T₀, T₁ and T₂ generation by selfcrossing and seeds collected in each generation.

Development of stable transgenic plants through tissue culture is stilla major problem in grain legumes so in an embodiment of presentinvention we have raised several independent transgenic shoots ofchickpea (T₀ generation) using explants.

Arabidopsis and tomato plants were grown on soilrite and normalautoclaved soil respectively. All plants were watered on alternate dayswith water or Hoagland solution. Chickpea shoots were grown on MS(Murashige and Skoog) medium containing respective hormones. Leaves oftransgenic Arabidopsis and chickpea plants from the progeny ofhomozygous lines and T₀ lines respectively were wounded by rapid pinpricks and kept for 5 minutes and 20 minutes and GUS expression wasmonitored by histochemical staining using glucuronic acid (1 mg/ml) in asolution containing phosphate buffer (pH 7.2) for its activity. Theleaves were kept for 3 hours for colour development in the presence ofcycloheximide (1.8 mM or 0.5 mg/ml) in an X-Gluc containing phosphatebuffer solution after 5 minutes and 20 minutes of wounding for colourdevelopment. Cycloheximide (an inhibitor of protein synthesis) was usedto ensure that the colour observed represented only the protein that wasproduced during the period of wounding. This ensured that whatever bluecolour was observed was due only to protein synthesized during the 5 minand 20 minute time course and not later during the development ofcolour. Wound inducible expression was seen from early stage ofdevelopment when seedlings were a few days old right up to when plantsstarted senescing. Expression was seen in stem, leaves, other parts ofplant (except roots) only upon wounding while in flowers abscission zoneexpression was found without wounding.

In another embodiment of the current invention the promoter was nexttested for universality of expression in different plants of economicimportance. Besides transgenic Arabidopsis (family Brassicaceae, arepresentative of Brassica/cabbage family), transgenic chickpea (familyLeguminaceae, a representative of the legume family) and transgenictomato (a representative of the Solanaceae family), other plants such ascotton (Malvaceae), tobacco (Solanceae), Rose (a representative ofRosaceae family) and Gladiolus (a representative of the monocot kingdom)were also transiently transformed by Agrobacterium inoculation through asyringe (containing the Agrobacterium transformed with the woundinducible promoter containing construct). For agroinjection, planttissues were kept for two days on the plant for integration of theAgrobacterium and the tissues then wounded by pin punctures.Histochemical GUS assay for checking GUS expression was performed asdescribed above. As shown in drawings strong wound inducible expressioncould be seen in transgenic Chickpea, Arabidopsis and Tomato leaves aswell as in agroinjected Cotton sepals, Rose petals, Gladiolus tepals andTobacco leaves only at the site of wounding. Transgenic plants wereraised with the use of CaMV35S constitutive promoter expression vectortaken as control to compare the efficacy of wound inducible promoter.

Further in another embodiment of this invention, to ensure thefunctionality of the promoter, transgenic Arabidopsis and Tomato plantswere raised using wound inducible construct having cry1Ac gene under thecontrol of RbPCD1 promoter by the same method (Agrobacterium mediated)as described above. The expression of the cryIAc gene (full length orC-terminal truncated) under the RbPCD1 promoter of sequence I.D.1 intransgenic Arabidopsis and Tomato plants led to normal plant developmentand seed set. Plants expressing RbPCDpro:cryIAcFL killed H. armigeralarvae feeding on these plants within 8 hours of release on the plantcompared to 24 hours in plants expressing the cryIAc gene under theCaMV35S promoter.

Thus, the transgenic plants were protected against insects by the woundinducible expression of controlling amount of insecticidal proteinCry1Ac. By controlling it is meant that a toxic (lethal) or combative(sub-lethal) amount of toxic protein is produced. At the same time, theplants are morphologically normal and may be cultivated in the usualmanner for human consumption should the Cry1Ac protein be deemed as safefor human health. In another embodiment of the invention, expression ofinsecticidal protein in plants (Arabidopsis/Tomato), was tested beforeand after wounding using an ELISA assay and found that the basal levelof expression of Cry1Ac is low (0.0049 ng/mm²) compared to 0.14 ng/mm²under the CaMV35S promoter. However within 5 minutes of wounding theexpression rises to 5 fold (6.28 ng/mm²) higher than in CaMV35S lines inlowest expressing transgenic lines. According to this aspect ofinvention, plants such as Arabidopsis/Tomato are provided that areinsect resistant due to presence in their genome of foreign DNAcomprising a DNA sequence encoding an insecticidal protein, undercontrol of a wound inducible promoter of sequence I.D.1 which ensuresexpression in wounded tissue. According to one embodiment of theinvention, expression of an insecticidal protein in the plants is suchthat, in the absence of wounding, the insecticidal protein is expressedat low or undetectable levels while upon feeding by insects, it isincreased rapidly (within 5 minutes) in the wounded tissue to a levelwhich is sufficient to kill the feeding insect. The present inventionfurther relates to use of the wound inducible promoter not only againstchewing insects but also against sucking pest provided a gene thatencodes a product toxic to sucking pests is used downstream of thepromoter.

Different methods such as sandwich ELISA (using Cry1Ac coated antibody)can be used to determine the wound induced expression of Cry1Ac proteinin transgenic plants of Arabidopsis/Tomato that were raised. Inparticular, according to the present invention the expression of Cry1Acprotein was checked in every puncture made by a forceps in a leaf discof unit area with a diameter of 1.5 cm upon wounding. For the presentembodiment of current invention, different number of punctures (2pricks, 4 pricks, 8 pricks and 12 pricks) of wounds (intended to meanmechanical damage) were made in the intact leaves of individualtransgenic lines and kept for different time intervals (5 minutes and 20minutes) following which the leaf discs were immediately frozen forprotein isolation. Unwounded transgenic leaf discs were taken ascontrol. Expression level of insecticidal protein is expressed as thepercentage of soluble protein as determined by antibody specificsandwich ELISA as described herein related to the total amount ofsoluble protein (as determined by Lowry method). An ELISA is useful inthe current invention for the correct estimation of wound induced Cry1Acprotein by one prick.

An aspect of present invention provides for a wound inducible expressionconstruct comprising RbPCD1 promoter of SEQ ID No. 1 or a complementarysequence thereof along with a gene of interest.

Another aspect of the present invention provides for a wound inducibleconstruct as herein described, wherein the gene of interest is selectedfrom the group comprising of insecticidal protein encoding genes againstsucking and chewing pests like cry1Ac (and various crystal proteingenes), anti-fungal genes like chitinases, genes encoding enzymes thatsynthesize low levels of medicinal compounds in plants.

Another aspect of the present invention provides for a wound inducibleconstruct as herein described wherein the promoter is activated within avery short time (5-20 minutes) of wounding.

Another aspect of the present invention provides for a a method ofpreparing wound inducible expression construct, said method comprisingthe steps of:

(a) isolating the RbPCD1 promoter gene;(b) amplifying RbPCD1 promoter gene of step (a) using primers selectedfrom, but not restricted to, GSP 1, and GSP 2 having SEQ ID No. 2, andSEQ ID No. 3;(c) obtaining a 523 nucleotide fragment of SEQ ID No. 1;(d) ligating the SEQ ID No. 1 of step (c) upstream of gene of interestin a expression cassette; and(e) inserting the expression cassette of step (d) in a plant expressionvector.

Another aspect of the present invention provides for a method ofpreparing wound inducible construct as herein described wherein the geneof interest is selected from group comprising of insecticidal proteinencoding genes against sucking and chewing pests like cry1Ac (andvarious crystal protein genes), anti-fungal genes like chitinases, genesencoding enzymes that synthesize low levels of medicinal compounds inplants Another aspect of the present invention provides for a method ofpreparing wound inducible construct as herein described wherein the geneof interest is expressed in both monocotyledonous and dicotyledonousplants.

Another aspect of the present invention provides for a transgenic plantcomprising of a wound inducible expression construct, wherein the woundinducible expression consist of RbPCD1 promoter having SEQ ID No. 1 or acomplementary sequence thereof along with a gene of interest.

Another aspect of the present invention provides for a the transgenicplant as herein described, wherein the gene of interest is selected fromthe group comprising of insecticidal protein encoding genes againstsucking and chewing pests like cry1Ac (and various crystal proteingenes), anti-fungal genes like chitinases, genes encoding enzymes thatsynthesize low levels of medicinal compounds in plants.

Another aspect of the present invention provides for a the transgenicplant as herein described, wherein the target proteins are expressedonly at the time of wounding.

Another aspect of the present invention provides for a the transgenicplant as herein described, wherein the promoter present in the woundinducible construct is activated within a very short time (5-20 minutes)of wounding.

Another aspect of the present invention provides for a method for makingtransgenic plant said method comprising the steps of:

(a) preparing a wound inducible construct comprising comprising RbPCD1promoter of sequence ID No. 1 or a complementary sequence thereof alongwith a gene of interest;(b) inserting the construct of step (a) in a Agrobacterium;(c) transforming the Agrobacterium of step (b) in the plants; and(d) obtaining a transgenic plant comprising and expressing a constructof step (a).

Another aspect of the present invention provides for a method for makingtransgenic plant as herein described, wherein the gene of interest isselected from the group comprising of insecticidal protein encodinggenes against sucking and chewing pests like cry1Ac (and various crystalprotein genes), anti-fungal genes like chitinases, genes encodingenzymes that synthesize low levels of medicinal compounds in plants.

Another aspect of the present invention provides for a method for makingtransgenic plant as herein described, whenever in the transgenic plantthe wound inducible construct is activated within a very short durationof wounding i.e. within 5-20 mins. of wounding.

Another aspect of the present invention provides for a method for makingtransgenic plant as herein described, wherein the transgenic plant hasthe ability to express any gene interest within a very short time ofwounding.

Another aspect of the present invention provides for use of a woundinducible expression construct comprising RbPCD1 promoter of sequence IDNo. 1 or a complementary sequence thereof along with a gene of interestuseful for early activation of wound inducible promoter.

Another aspect of the present invention provides for use of a woundinducible expression construct comprising RbPCD1 promoter of sequence IDNo. 1 or a complementary sequence thereof along with a gene of interestfor preparing transgenic plants.

Another aspect of the present invention provides for use of a woundinducible expression construct as herein described, wherein thetransgenic plants expressing gene of interest within short time ofwounding (within 5-20 mins. of wounding).

Another aspect of the present invention provides for use of transgenicplant for expression construct comprising RbPCD1 promoter of sequence IDNo. 1 or a complementary sequence thereof along with a gene of interest.

Another aspect of the present invention provides for use of transgenicplant as herein described, wherein the transgenic plant expresses geneof interest within short time of wounding (within 5-20 mins. ofwounding).

Another aspect of the present invention provides for the use of novelwound inducible constructing for preparation and/developing transgenicplants, wherein the said method involves (a) preparing a wound inducibleconstruct comprising comprising RbPCD1 promoter of sequence ID No. 1 ora complementary sequence thereof along with a gene of interest; (b)inserting the construct of step (a) in a Agrobacterium; (c) transformingthe Agrobacterium of step (b) in the plants; and (d) obtaining atransgenic plant comprising and expressing a construct of step (a).

Another aspect of the present invention provides for use of novel woundinducible, wherein the wound inducible construct enable immediate andfast expression of gene of interest. The wound inducible promoter isactivated within 5-29 mins of physical or chemical wound injury and itis because of which the gene of interest also present in the constructgets activated. Thus the present invention in its one of the embodimentalso provides the use of wound inducible promoter for preparingtransgenic plants capable of fast or immediate expression of gene ofinterest in the transgenic plants when they receive a chemical ormechanical or biological wound.

EXAMPLES

The present invention will now be more fully described with reference tothe accompanying examples and drawings. It should be understood,however, that the following description is illustrative only and shouldnot be taken in any way as a restriction on the generality of theinvention described above.

The following non limiting examples describe the construction ofsuitable chimeric foreign gene including a DNA sequence encoding aninsecticidal protein under the control of early wound inducible promoterfor the expression of insecticidal protein under it and obtaining aresistance against Helicoverpa armigera. Unless stated otherwise inexamples, all techniques are carried out according to standard protocolsas described in Sambrook and Russell Molecular cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, NY, involumes 1, 2 and 3 of Ausubel et al., (1989).

Example 1 Preparation of Genome Walking Library & Isolation of RbPCD1Promoter of Sequence I.D.1: Preparation of Genome Walking Library:

The genome walking library of rose was constructed from rose petal DNAdigested with DraI, EcoRV, PvuII, StuI and SmaI using standard protocolsof BD Genome Walker Universal Kit (BD Biosciences Clontech, USA). Inbrief, 2.5 μg genomic DNA each was subjected to digestion with fivedifferent restriction enzymes such as DraI, EcoRV, PvuII and StuI andSmaI, in separate 1.7 ml microfuge tubes. Tubes were kept at 37° C.overnight and a small proportion was checked on 0.6% agarose gel toensure proper digestion of DNA. Subsequently, reaction mixtures wereextracted with phenol: chloroform and precipitated by adding 1/10 volumeof 3M sodium acetate (pH 4.5), 2 volume of ice cold 95% ethanol. DNAfrom each tube was pelleted by centrifugation at 15,000×g for 10 min,the pellet was washed with 100 μl of ice cold 80% ethanol, vacuum driedand suspended in 20 μl of TE (Tris 10 mM, EDTA 1 mM, pH 8.0). From eachtube, 4 μl was taken in separate tube and 1.9 μl of Genome walkeradapter (25 μM), 0.8 μl of 10× ligation buffer and 0.5 μl of T4 DNALigase (6 U/μl) and 0.8 μl of H₂O were added in each tube separately andincubated at 16° C. overnight. The ligation reaction was stopped byplacing all the tubes at 70° C. for 5 min.

Isolation of RbPCD1 Promoter of −523nt of Sequence I.D.1: Primary PCR:

Primary PCR was performed using Genome-walker library of rose with AP1as adapter primer (designed in vitro) and GW-GSP1 primers of RbPCD1. Inprimary PCR, components were added as follows: sterile water 30 μl,genome-walker library 4 μl (containing a mixture of all 5 libraries),10×PCR buffer 51 μl, 10 mM dNTP 5 μl, 12.5 picomoles each of AP1 andGSP1 primers (2.5 μl each) and (1 U/μl) Klen Taq DNA Polymerase 1 μl.The PCR was initiated by 7 cycles of denaturation step at 94° C. for 2sec and extension at 72° C. for 3 min, followed by 32 cycles ofamplification each consisting of denaturation at 94° C. for 2 sec,annealing at 67° C. for 3 min followed by a final extension at 67° C.for 4 min.

Secondary PCR:

For secondary amplification, 5 μl of the amplified reaction of primaryPCR was diluted to 500 μl with sterile MQ H₂O, and 2 μl of this was usedas template for secondary PCR amplification. The reaction conditionswere as above, except that instead of GSP1 and AP1, the primers usedwere GSP2 (a gene specific reverse primer internal to GSP1) and AP2 (atruncated adapter primer). The PCR was initiated by 5 cycles ofdenaturation step at 94° C. for 2 sec and extension at 72° C. for 3 min,followed by 20 cycles of amplification each consisting of denaturationat 94° C. for 2 sec, annealing at 67° C. for 3 min followed by a finalextension at 67° C. for 4 min. Amplified fragments were resolved onagarose, excised, purified and cloned in pTZ57R/T using the InsT/Acloning kit from Thermo scientific and sequenced.

Example 2 Preparation of Wound Inducible Construct:

Amplified −523 nt fragment of RbPCD1 promoter of sequence I.D.1 wasprimarily cloned into pTZ57R/T [InsTAclone PCR cloning kit, Cat. No.#1241, Thermo scientific, Lithuania (EU)] and sequenced, then sub clonedinto the pBI101 vector (a promoter less binary vector—Clontechlaboratories, USA; Cat No 6017-1) upstream of the beta glucuronidasegene (GUS-gene present within pBI101, from Clontech laboratories, USA,Cat No 6017-1) at the SalI site. In another construct, the amplified 523nt fragment of RbPCD1 promoter was cloned in HindIII digested pTZ57R/Tand then sub cloned at HindIII restriction site upstream to full lengthcry1Ac gene (obtained with permission from Dr PK Singh/RK Tuli's lab,CSIR-NBRI) in the plant expression vector pBI101 (FIG. 1). The resultantpBI101 carrying the wound inducible chimeric expression cassette wastransformed into Arabidopsis, tomato and chickpea via Agrobacteriumtumefaciens strain GV3101 (strain GV3101pMP-90RK from DNA CloningServices, Germany, Cat No 1004).

Example 3 Process of Making Transgenic Arabidopsis, Tomato and Chickpea:Transformation of Arabidopsis Plants by Floral Dip Method

Arabidopsis (Arabidopsis Biological Resource Centre, Ohio StateUniversity, USA) plants were grown and when the secondary bolts startedto flower, plants were transformed with recombinant plasmid constructsby floral dip method as described by Clough and Bent (1998). For this asingle colony from plate containing Agrobacterium tumefaciens GV3101strain carrying gene of interest was picked and inoculated in 5 ml LBmedium supplemented with antibiotics rifampicin (50 μg/ml), kanamycin(50 μg/ml) and gentamycin (30 μg/ml). The primary culture was grown at28° C., 220 rpm for 24 h. From this 1 ml culture was inoculated in 500ml of LB medium containing the same set of drugs and grown for 24 hoursat 28° C., 220 rpm. Cells were harvested by centrifugation at 5000×g for5 min at room temperature. Pellet was resuspended gently in 5% sucrosesolution to a final OD₆₀₀ of approximately 1. Prior to dipping, thedetergent Silwet L-77 (Lehle Seeds, USA) was added in Agrobacterium cellsuspension prepared in 5% sucrose, to a final concentration of 0.05% andmixed gently by swirling the container. This solution was termed asinfiltration medium. The floral inflorescences were dipped in theinfiltration medium for 10 to 15 sec with gentle swirling. A film ofliquid coating the plant was seen and excess liquid was wiped off.Dipped plants were placed in dark under a dome or cover for 16 to 24 hto maintain high humidity and then back in the racks and subjected tonormal light. For a higher rate of transformation, the same plants withnewer flowers were again dipped in a fresh suspension of recombinantAgrobacterium. Plants were allowed to grow and moisture of soil wasmaintained with nutrient medium till all the siliques had grown fully.As the siliques began to dry, watering of plants was stopped and seedswere allowed to become mature. The dried seeds were harvested, separatedfrom other dried tissues and stored at room temperature in dry place.The dried seeds/transformants were selected using antibiotic Kanamycin(50 mg/ml) on half MS agar plate as selection marker. Transformants wereidentified as Kanamycin resistant seedlings that produced green leavesand well established roots in the selection medium. Viable seedlingswere transferred into the individual pots filled with soilrite saturatedwith nutrient media. Pots were kept in plastic trays and the plants wereleft to grow in normal conditions. Transformed plants were furtherconfirmed by PCR with gene specific primers using genomic DNA astemplate.

Agrobacterium Mediated Genetic Transformation of Tomato Plants:

Tomato seeds (National Seed Corporation Ltd., Pusa Campus, New Delhi)were surface sterilized with 50% sodium hypochlorite (bleach) containing0.1% Tween-20 in a laminar flow hood for 5 mins. After that seeds werewashed thoroughly with sterile Milli-Q water for 4-5 times. The surfacesterilized seeds were blotted on Whatmann filter paper and allowed togerminate in seed germination medium (half strength MS medium) underculture room conditions at 25° C. Tomato transformation was carried outusing 1 month old plants having large number of expanded leaves. Youngexpanded leaves were excised and several small discs cut out withsterile blade in order to increase infection opportunity ofAgrobacterium and to enhance transformation efficiency. The leaves wereplaced in MS medium, precultured for 24 to 48 h at 25° C. under lowlight conditions (10 μEm−2s−1). On the same day a single colony ofAgrobacterium containing the construct was inoculated in 5 ml LB/YEBmedium with appropriate antibiotics for 16-18 h at 28° C. 50 μl of thisculture was used to inoculate bacteria in 25 ml YEB with antibiotics(Rifampicin+ Gentamycin and Kanamycin) and allowed to grow to an O.D.0.2-0.4 at 600 nm. The bacterial culture was centrifuged at 5000 rpm for5 min at 4° C. The pellet was resuspended in liquid half strength MSbasal medium and acetosyringone was added at a concentration of 200 μMto enhance the infection efficiency. Pre-incubated leaf explants wereco-cultivated with Agrobacterium culture (8-10 ml) for 25 min withoccasional swirling. The explants were then dried on sterile Whatmannpaper and incubated in co-cultivation medium in culture room in dark for2 days. Explants were then transferred onto plates containing MS mediumsupplemented with 0.1 mg/l IAA+1.0 mg/l BAP+1.0 mg/l Zeatinriboside or0.1 mg/l IAA+1.0 mg/l Zeatinriboside+50 mg/l Kanamycin and 500 mg/mlcefotaxime for 6 to 7 days to eliminate Agrobacterium contamination andsupport explant growth. The explants were kept for callusing and shootinitiation on the same medium in culture room conditions for 2-3 weeks.At the time of third selection calli were transferred for shooting byincreasing auxin concentration and lowering down cytokinin concentration(0.5 mg/l IAA+0.25 mg/l Zeatinriboside) for selection of elongatedtransformants. Elongated shoots were then transferred to rooting mediumhaving 25 mg/l kanamycin and cefotaxime for 12-15 days. Rooted plantswere both hardened in KNOPS solution for 1 week as per standard protocolor directly transferred to soil and finally shifted to glass house foroptimum growth.

Agrobacterium Mediated Genetic Transformation of Chickpea Plants:

The mature healthy seeds of chickpea (C. arietinum L.—CSIR-NBRI field)were surface sterilized (0.01% HgCl₂ and dipped 30 sec in 70% ethanolfollowed by washing with 3-4 times H₂O) and incubated for germination inculture medium, consisting of Murashige and Skoog (MS) salts, B5vitamins, 3% (w/v) sucrose, 0.8% (w/v) agar and 1.0 mg/l6-benzylaminopurine (BAP). The cultures were incubated in the cultureroom maintained at 24±1° C. under cool white light of intensity 60 mmolm⁻²s⁻¹ for 16 h photoperiod. A single positive colony from platecontaining Agrobacterium tumefaciens GV3101 strain carrying a chimericwound inducible cassette of interest was picked and inoculated in 5 mlYEB medium supplemented with antibiotics rifampicin (50 μg/ml),kanamycin (50 μg/ml) and streptomycin (30 μg/ml). The primary culturewas grown at 28° C., 220 rpm for 24 h. From this 50 μl culture wasinoculated in 25 ml of YEB medium containing the same set of drugs andgrown for 24 hours at 28° C., 220 rpm and harvested, the bacterialpellet was resuspended in liquid MS medium to obtain an OD₆₀₀ nm between0.8 to 1.0. Various cotyledonary nodes were excised with sterilescalpel, sonicated for 30-60 sec, vacuum infiltrated for 5 min andinfected in Agrobacterial suspension for 20 min with constant shaking at75 rpm 24° C. The cotyledonary nodes were blotted on sterile filterpaper and incubated on semi-solid MS medium supplemented withappropriate plant growth regulators and incubated in dark for 2 daysunder culture room conditions. After co-cultivation for a period of 2-4days, the cotyledonary nodes were transferred to Petri dishes having MSmedium supplemented with required plant growth regulators (PGRs) and250-500 mg l⁻¹ cefotaxime and cultured one week under normal growthconditions. The cotylednary nodes were sub-cultured for two week onmedium supplemented with 50 or 100 mg l⁻¹ kanamycin along with theappropriate amounts of PGRs and 100-250 mg l⁻¹ cefotaxime. The emergingshoots from the cotyledonary nodes cultured on kanamycin-supplementedmedium for 3 successive cycles for screening of transformants. Shootsobtained were clonally propagated on shoot elongation medium, prior torooting or grafting. The rooted or grafted plant stocks were processedfor hardening and acclimatization, before their transfer to glasshousefor further growth, development and molecular characterization. To testthe transgene integration, DNA was isolated from the small piece oftissue taken from shoots and PCR reaction performed by using respectiveprimers.

Example 4 Histochemical Analysis of Transgenic Lines for TransgeneIntegration

Histochemical GUS staining was carried out as described by Gattolin etal., (2006). Tissue samples of different plants (All plants—obtainedfrom CSIR-NBRI garden except Arabidopsis and tomato) were incubated in 1mg/ml X-gluc (Biosynth AG, Switerland) solution containing 50 mM sodiumphosphate buffer pH 7.2, 0.5 mM K₃Fe (CN)₆ and 0.5 mM K₄ Fe (CN)₆ for16-24 h at 37° C. After incubation tissues were destained in 70% ethanolat 37° C. and stored until examination. Light microscopy was performedon a Leica Wild M3Z microscope (Leica Germany). As shown in drawings,the histochemical GUS assay of the RbPCD1 promoter showed strong woundinduction in response to mechanical wounding as well as insect wounding(Helicoverpa armigera and aphid-obtained from chickpea fields ofCSIR-NBRI and raised in entomology lab) in transgenic Arabidopsis leaves(FIGS. 2 a & b). Apart from Arabidopsis, a strong wound inducibleexpression could be seen in transgenic chickpea as well as inagroinjected cotton sepals, rose petals, Gladiolus tepals and tobaccoleaves only at the site of wounding (FIG. 2d ). However, a uniform GUSexpression throughout the whole plant including leaf could be seen intransgenic plants expressing GUS gene under the CaMV35S promoter inplants transformed with expression vector pBI121 (pBI121, GUS driven byCaMV35S promoter—Clontech laboratories, USA 0018-1) and used as controlto compare the efficacy of wound inducible promoter (FIG. 2c ).

In order to examine the spatiotemporal expression patterns ofRbPCD1pro::GUS, whole plants at different developmental stages werehistochemically stained. A strong wound inducible expression was seenfrom early stage of development when seedlings were a few days old rightup to flowering stage while wound inducible expression decreased whenplants started senescing. Expression was seen in stem, leaves, otherparts of plant (except roots) only upon wounding while in flowerabscission zones expression was found without wounding (FIG. 2e ).

Example 5 ELISA Assay of Transgenic Lines and Insect Mortality Assay(DAS-ELISA) (Double Antibody Sandwich Enzyme Linked Immunosorbent Assay)

Quantitative estimation of protein produced in transgenic Arabidopsisand tomato plants was performed using a Double Antibody Sandwich EnzymeLinked Immunosorbent Assay (DAS-ELISA-Agdia Inc USA, Cat No PSM14900/0480) with pre-antibody coated wells in microtitre plates. Tocheck the expression of Cry1Ac protein under the wound induciblepromoter which expresses within 5-20 minutes, intact leaves oftransgenic plants were wounded with a pair of forceps (1 mm²) by rapidpricking and after that leaf discs were made by using chopper ofdiameter size approximately 1.5 cm. Control (unwounded) leaf discs wereimmediately frozen and wounded leaf disc tissues taken 5-20 minutesafter wounding followed by protein isolation using protein extractionbuffer. For protein isolation, leaf discs were crushed in liquidnitrogen and PBST [NaCl-8.0 g, sodium diphosphate, dibasic(anhydrous)—1.15 g, potassium phosphate monobasic (anhydrous)—0.2 g,potassium chloride—0.2 g, Tween-20—0.5 g, pH 7.4 (dissolved in 900 mldouble distilled water)] and kept at 4° C. for 10 min. Extraction bufferalong with the protein extract was centrifuged at 12000×g for 10 min at4° C. Supernatent was taken and added to the kit wells of 100 μl and kitantibody of 100 μl added and incubated at 37° C. for 2 h. This was thenwashed with PBST buffer for 5-6 times. To this, 100 μl of peroxidasesubstrate solution was added and incubated at 37° C. for 10-15 min andread at 650 nm. To calculate the amount of Cry1Ac protein expressed perwound the respective ODs were plotted against Standard Cry Plot.

Example 6 Transient Expression of Promoter by Agroinjection to Test itsEfficacy in Other Plants

The promoter fragments, cloned in pBI101 upstream to GUS were used todetermine the expression. These promoters were transiently expressed indifferent plants by agro-infiltration. Agro-injection was performed asdescribed by Orzaez et al. (2006) with few modifications. In brief, asingle colony of Agrobacterium strain GV3101 harbouring the promoterconstruct was inoculated in 5 ml LB medium containing selectiveantibiotics rifampicin (50 mg/ml), kanamycin (50 mg/ml) and gentamycin(30 mg/ml) and grown overnight at 28° C. at 200 rpm. From this culture,1 ml was transferred to 50 ml induction medium (LB medium supplementedwith 0.5% Sucrose, 2 mM MgSO₄, 20 mM acetosyringone, 10 mM MES, pH 5.6plus same set of antibiotics) and grown overnight at same condition toan OD₆₀₀ of 1.4. Cells were recovered by centrifugation at 5000×g for 5min at RT, resuspended in infiltration medium (10 mM MgCl₂, 10 mM MES,200 mM acetosyringone, pH 5.6; optical density 1.0) and incubated atroom temperature with gentle agitation (50 rpm) for a period of 2 h. Foragro-injection, control rose flowers (with only two or three petals fromthe outer whorl open), gladiolus tepals, cotton sepals and tobaccoleaves were selected and a needle (size 23, dimensions 0.63325 mm)fitted on to a 2 ml syringe (filled with the 0.5 ml recombinantagrobacterial suspension containing 0.01% acetosyringone) was lightlyinserted in the bottom of the petals, sepal and through mid rib of thetobacco leaves. The agrobacterial suspension was slowly forced into theRose petals, gladiolus tepals, tobacco leaves, cotton sepals and allowedto infiltrate all through the respective tissue up to the point ofattachment of the petal/tepal with the thalamus and of the leaf with thestem. Agroinjection was performed with three flowers per construct andthree petals per flower and three leaves per plant. The excesssuspension was wiped off and the buds were kept for 2 days on the plant.As a control, Agrobacteria containing pBI101 (no promoter), pBI121 (GUSdriven by CaMV35S promoter), were also used for agro-infiltration ofindependent tissues. After two days from agro-infiltration,flowers/sepals/tepals/leaves were punctured with pair of forceps andcarefully cut out under water. After wounding, the petals, tepal, sepalsand leaves were detached and stained for GUS expression as describedabove.

Example 7

To check the effectiveness of putative transgenic (Arabidopsis andTomato) plants expressing cry1Ac under the control of wound inducibleRbPCD1 promoter against insect attack, detached leaf as well as wholeplant insect bioassays were conducted with different larval stages ofinsect Helicoverpa armigera. Toxicity of transgenic plants due toexpression of Cry1Ac protein for insect was analyzed by conducting nochoice insect bioassays on detached leaf as well as whole plant on T₁and homozygous (T₃) lines of Arabidopsis while primary transformants(T₀) and T₁ generation plants were selected for tomato. Comparison ofcry1Ac expression and mortality in transgenic plants were performedagainst cry1Ac expressed under constitutive CaMV 35S promoter intransgenic Arabidopsis plants and wild type Col-0 used as control. Fortomato plants, non-transformed wild type plants were used as control. Asshown in drawings, the result of the insect bioassay showed that thewound inducible expression of cry1Ac in transgenic Arabidopsis andtomato conferred complete protection against Helicoverpa armigera incomparison to non-transgenic control plants (FIGS. 3 & 4).

Advantages/Uses

The promoter has several advantages over the other known promoters suchas CaMV35S that is used for constitutive expression of insecticidaltoxin proteins.

-   1. It will increase the efficacy of several insecticidal toxin    protein genes that are currently in use for controlling Lepidopteran    insects by ensuring low level expression of the toxic genes during    normal plant growth but much higher level expression (as compared to    the CaMV35S promoter) at the time of insect wounding. The low level    expression under non wounded conditions during normal plant growth    (in absence of insect attack) would prevent the deleterious effects    of the high levels of toxic protein on whole plant development (seen    under the CaMV35S promoter) and ensure normal plant development and    higher yields.-   2. The promoter would ensure high level expression of the protein    even after wounding at the flowering and late fruiting stages    (beyond 90 days (as evident from expression in cotton sepals) which    is currently not possible in maize and cotton due to inherent low    level expression of the CaMV35S promoter at the reproductive stages.-   3. The promoter would be effective against both chewing and sucking    pests since it is activated by wounding by both these types of    insects. It thus has a wide canvas for controlling damage by biotic    stress.    -   Examples: Cotton, chickpea and other legumes, Brassica family        members (like Arabidopsis), Solanaceae family members like        tomato, tobacco etc, monocots like Gladiolus.-   4. The control of the insecticidal gene expression only under    wounding would ensure normal plant development and may allow several    other plants, which are currently not so easily transformed due to    insect protein toxicity, to be transformed.-   5. It will reduce the load on the plant by synthesizing the toxic    protein only at the time of insect infestation/wounding and not at    all times when it is not needed.-   6. It would be useful for rapid and high level expression of    valuable proteins in a wound inducible manner.-   7. It would be useful for rapid expression of any medicinal or    valuable chemical that requires prior action by a protein or enzyme.

1. A wound inducible expression construct consisting of the nucleotidesequence of Sequence ID No. 1 or the complementary sequence of it alongwith the gene of interest useful for early activation of wound induciblepromoter.
 2. The construct as claimed in claim 1 wherein the gene ofinterest is selected from the group comprising of insecticidal proteinencoding genes against sucking and chewing pests like cryIAc (andvarious crystal protein genes), anti-fungal genes like chitinases, genesencoding enzymes that synthesize low levels of medicinal compounds inplants.
 3. A method for preparing the construct as claimed in claim 1comprising of steps: I. providing RbPCD1 gene having seq id no. 1; II.amplifying seq id no. 1 using primers selected from, but not restrictedto, GSP 1, and GSP 2 having Sequence ID No. 2, and Sequence ID No. 3;III. ligating the gene of interest downstream to the promoter; IV.cloning the sequence obtained in step (iii) in suitable vector for plantexpression
 4. The wound inducible promoter as claimed in claim 1 whereinthe promoter gets activated within 5 minutes of wounding.
 5. A processfor making transgenic plants comprising wound inducible promotercontaining construct as claimed in claim 1 wherein the process comprisestransforming the expression construct as claimed in claim 1 into theplant using Agrobacterium mediated transformation.
 6. The process asclaimed in claim 5 wherein the gene of interest is selected from thegroup comprising of those encoding insecticidal proteins, enzymes,antibodies, therapeutic proteins or any protein responsible forproducing a valuable chemical comprising wounding of transgenic plants.